The purpose of this project is to determine second messenger pathways involved in receptor regulation of neurotransmitter levels. Using bovine adrenal medullary cells, Dr. Raimo Tuominen in our lab had found that angiotensin II (AII) and nicotine increased protein kinase C (PKC) translocation and diacylglycerol (DAG) levels in the cells. As this is the only second messenger pathway which our lab has shown to be increased long-term, this project was continued and expanded. Following C 3 H]choline and [3 H]inositol incorporation and breakdown in these cells, it appears that phosphatidylcholine is the likely source of long-term (10 minutes or later) DAG formation. Using [3H]phorbol libutyrate binding to follow PKC translocation, agonists which stimulate Ca2+ influx rather than intracellular Ca2+ mobilization were found to be more efficacious in stimu- lating [3 H]PdBu binding in BAM cells. While Ca2+ mobilizing pathways are distinct for different agonists, it is unclear whether the DAGs formed and the PKCs translocated are the same for each agent. AII stimulates C 3 H]PdBu binding through an extracellular Ca2+- and omegaCgtx-sensitive pathway, but the effect is small and transient in contrast to the effect of nicotine and depolarizing stimuli. The same picture emerges with [3H]PdBu binding to C6 glioma cells, SMS-KCNR neuroblastoma cells and primary cultures of hippocampal and striatal cells (other cells in which enkephalin MRNA levels may be regulated by Ca2+ and/or PKC): extracellular Ca2+ appears much more important for PKC translocation than is intracellular Ca2+ mobilization. An interesting finding resulting from this technique was that dopamine increased [3 H]PdBu binding in striatal cells through a novel D-1 receptor, which was almost as effective as well-characterized Ca2+-Mobilizing agonists such as norepinephrine and excitatory amino acids. The dopamine response was antagonized by delta and mu (but not kappa) opiate receptor agonists; it is unclear whether this antagonism is specific to PKC activation and [3H]PdBu binding.